The long-term goal of this project is in-depth systems-level understanding of G proteins signaling network and its physiological consequences. In the coming term this focus continues with the goal of translating mechanistic understanding into therapeutics. During the current term we used multivariate experiments and network building and analyses to get an initial understanding of the design logic underlying receptor regulated neurite outgrowth. We have found that experimentally identified agents that promote neurite outgrowth in cultured cells work on primary neurons in microfluidic chambers and also promote axonal regeneration in vivo. The findings using agonists that activate the cannabinoid-1 (CB1)receptor-Go/i and interlukin-6 (IL60 receptor-JAK-STAT3 network suggested that there may be many components in the regulatory networks involved in subcellular processes within the cell that could control neurite outgrowth and axon regeneration. To obtain an unbiased view of the changes triggered by receptor activation we profiled mRNA changes by mRNA-Seq and protein changes by mass-spectrometry. We used components that changed as seed nodes to build functional direct interaction networks and analyze control patterns. Our initial analysis indicates a pattern of deep control wherein components and pathways involved in core subcellular functions such as cell surface protein deglycosylation, membrane biosynthesis & vesicle transport, cell adhesion and actin filament dynamics play critical roles in neurite outgrowth. These preliminary observations give rise to the hypothesis that understanding CB1R and IL6R signaling network dependent deep control at the level of subcellular functions will allow us to identify drug targets and drug combinations that can be used to obtain restoration of function by axon regeneration after injury. The systems level approach we propose combines network analyses with dynamical modeling is based on both large-scale profiling as well as small-scale functional assays. It will be geared to determine What; Where; When: what drugs can be used; where (cell body, growing axon shaft or growth cone) should the drugs be applied; and when should the drugs be applied. We have 3 aims: Aim 1: Determine the subcellular processes involved in neurite/axon outgrowth by obtaining a genome-wide view of the changes associated with CB1R and IL6R network triggered changes in Neuro2A cells and rat primary cortical neurons using a combination of high throughput experiments and development and analysis of networks using graph theory. Aim 2: Validate the multiple subcellular mechanisms underlying CB1R and IL6R triggered axon outgrowth by functional assays in primary cortical neurons in microfludic chambers and develop multicompartment ODE and hybrid dynamical models of axon outgrowth to develop predictions of what drugs can be used in combination with HU-210 and IL6, where should they be applied and when should they should be applied. Aim 3: Use the optic nerve injury model in rats to test if application of a combination of drugs as determined by the experiments and network & dynamical models can restore function in vivo.